This study represents the first investigation demonstrating the contractile response to exogenous acetylcholine (ACh) in the isolated human vas deferens. Pharmacological characterization of cholinergic receptors was achieved using selective antagonists to define receptor subtypes. In the HVD the effect of exogenous ACh is revealed as a dose-dependent sudden increase in the basal tension of the vasa. The ACh receptors of the HVD were competitively antagonized by atropine (ATR) with a high pA2 value (8.78). The main finding of this study is the presence of cholinergic receptors of the pharmacologically defined M1-ACh subtype in the isolated HVD, according to the pA2 values obtained with pirenzepine (PRZ) 7.39, AF-DX 116 (AF) 5.92 and 4-DAMP 5.65, M1-ACh, M2-ACh and M3-ACh selective antagonists, respectively. Prazosin (PZ), a selective alpha 1-adrenergic antagonist, displayed a similar competitive antagonism for the contractile response evoked both by ACh (pA2 = 8.69) and NE (pA2 = 8.58) in the HVD. The antagonism exerted by PZ on the ACh-induced contractile response of the HVD, suggests that ACh probably acts at a presynaptic level stimulating the release of NE from an adrenergic neuron. According to these findings, the receptor involved in this action, located in the proximity of the nerve terminals, seems to be of the M1-ACh subtype.